Meterodimeric T lymphocyte receptor antibody

ABSTRACT

Disclosed is a heterodimeric T lymphocyte receptor comprising an alpha and a beta subunit. Each subunit consists of a signal peptide, variable, joining, constant, transmembrane, and cytoplasmic regions. The two subunits are connected by a disulfide bond between cysteine residues located between the constant and transmembrane region. The structure, amino acid, and nucleotide sequence of the lymphocyte receptor were determined using cDNA cones derived from a functional murine cytotoxic T lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and have significant sequence homologies to immunoglobulin V and C genes. Both the T cell receptor protein and its subunits may be produced from the cDNA clones. The protein molecules may be further used for the production of T-cell clone specific antibodies.

The U.S. government has rights in this invention by virtue of Grant No.NIH-5-POl-CA28900-04, NIH-5-P30-CA14051-13 and the Arthritis Foundation.

REFERENCE TO RELATED APPLICATION

This application is a divisional to U.S. Ser. No. 666,988 filed Oct. 31,1984 entitled "Heterodimeric T Lymphocyte Receptor" by Haruo Saito,David M. Kranz, Herman N. Eisen, and Susumu Tonegawa which is acontinuation-in-part of our co-pending patent Ser. No. 620,122, now U.S.Pat. No. 4,873,190, filed Jun. 13, 1984, entitled "T Lymphocyte ReceptorSubunit".

BACKGROUND OF THE INVENTION

The vertebrate immune system is characterized by its ability to respondto an enormously diverse set of antigenic determinants. This capabilityis due to the synthesis by the body of a set of glycoproteins whosespecificity for a single antigen is determined by a variable sequence ofamino acids which binds to the antigen. The glycoproteins, whichrecognize and bind free antigens, are produced by B cells and are calledimmunoglobulins (Ig).

Each B cell, or bone marrow-derived lymphocyte, produces antibodyspecific for only one antigen. It has been theorized that the type ofimmunoglobulin which is produced by the B cell is generated by a seriesof gene rearrangements and RNA splicing events that result inpolypeptide chains consisting of variable and constant regions. Theseregions can be subdivided into domains held together by interchain andintrachain disulfide bridges situated at the same relative positions.The characteristic primary and secondary structure is made up of heavyand light chains which begin with a leader peptide of 17-29 residues,followed by a variable (V) region of 94-97 residues, then a joiningregion of 13-17 residues (J), then a constant region (C). The domains ofthe constant regions of the immunoglobulins are encoded by separateexons from those for the variable region and do not appear to rearrangeduring development.

T cells or thymus derived lymphocytes, like B cells, are capable ofrecognizing a wide range of different antigens. The ability to recognizea given antigen is also fixed in any particular clonal line of T cell. Tcells, however, recognize only antigens located on the surfaces of cellsin the specific molecular context of self major histocompatabilitycomplex (MHC) gene products, not freely circulating antigens. T cellreceptors recognize foreign antigens (such as viral antigens) in themolecular context of the T cell host's self-MHC gene products orrecognize foreign MHC gene products. Cell surface antigens include tumorcell and viral antigens. The ability to recognize cell-bound antigens isacquired when the T cells differentiate in the host thymus.

Effective antisera and monoclonal antibodies have now been developedwhich recognize and precipitate clone-specific proteins on the surfaceof functional T cell clones, hybridomas or T cell tumors. Studies usingthese antibodies have suggested that the specificity-determining portionof a T cell receptor is a heterodimeric glycoprotein of about 90,000daltons and consisting of an alpha subunit and a beta subunit heldtogether by an inter-chain disulphide bond(s). Peptide fingerprintanalysis of alpha and beta chains from several T-cell lines suggest thatboth subunits are composed of variable (V) and constant (C) regions.

Three groups of workers have since succeeded in isolating Tcell-specific cDNA (complementary DNA) clones of mouse or human originwhich are homologous to immunoglobulin genes. S. M. Hedrick, E. A.Nielsen, J. Kevaler, D. I. Cohen, and M. M. Davis, as reported inNature, 308, 153-158 (1984), using a mouse, antigen specific,MHC-restricted T helper hybridoma TM86, demosstrated that the cDNAencoded a protein composed of an amino-terminal variable and acarboxy-terminal constant region. They also showed that thecorresponding genomic DNA sequences had undergone clone-specific somaticrearrangements in various T cell lines.

Y. Yanagi, Y. Yoshikai, K. Leggett, S. P. Clark, I. Aleksander, and T.W. Mak, in Nature, 308, 145-149 (1984), reported the nucleotide sequenceof a cDNA clone YT35 derived from the human leukaemic T cell line,MOLT-3. The predicted amino acid sequence encoded by the human cDNAclone of Yanagi et al. is highly homologous in the constant region tothe cDNA clone isolated by Hedrick et al.

We recently reported in Nature, 309, 757-762 (1984) the cloning andsequencing of two related but distinct cDNA clones pHDS4/203 and pHDS11from a functional murine cytolytic T lymphocyte clone, 2C, the teachingsof which are incorporated herein. The genes corresponding to these cDNAclones are rearranged and expressed in T cells (and not in other cellsthat have been examined such as B cells, kidney cells, etc.) and alsohave significant sequence homologies to immunoglobulin V and C genes.The amino acid sequence encoded by clone pHDS11 corresponds to the sameT-cell receptor genes as those isolated by Hedrick et al and Yanagi etal.

A second cDNA clone pHDS4/203 was identified as the gene coding for thealpha chain of the T cell antigen receptor. However, subsequent studieshave shown that this clone lacks sequences that correspond to sites forN-linked glycosylation and it has recently been shown that both thealpha and beta chains of the T cell receptor are N-glycosylated. Despitethe fact that the four clones: YT35, isolated by Yanagi et al, TM86,isolated by Hedrick et al, and pHDS11 and pHDS4/203, isolated by Saitoet al, share several common properties, it is unlikely that the clonepHDS4/203 encodes for the alpha subunit of the T cell receptor proteincomprising the beta subunit encoded by the genes YT35, TM86, and pHDS11.These four cDNA clones share the following common properties. They areexpressed in T cells but not in B cells. The corresponding genes arerearranged in T cells and not in B cells. In the encoded proteins, thereare distinctive regions that, proceeding from amino- tocarboxy-terminus, correspond to a signal peptide, twoimmunoglobulin-like domains, a transmembrane peptide rich in hydrophobicamino acids, and a short cytoplasmic peptide. Their deduced amino acidsequences have low but significant homology to those ofimmunoglobulin-chains. Each gene is composed of separate V, J and C genesegments. In the cases of YT35 and TM86, the corresponding genes havealso been shown to have D segments. Their V, (D), and J segments havecharacteristic signal sequences for gene rearrangement (heptamer andnonamer separated by either 12 or 23 base pairs).

Due to the substantial homology between the sequences of TM86, YT35 andpHDS11, it is likely that they represent the beta subunit of the T cellreceptor.

Due to the absence of N-glycosylation sites on the gene PHDS4/203 Saitoet al, it is unlikely that this gene represents the alpha subunit of theT-cell receptor.

It is therefore an object of the present invention to provide a T-cellreceptor gene or nucleotide sequence which codes for both subunits ofthe T-cell receptor.

It is a further object of the present invention to provide murine cDNAclones which code for either the alpha or beta subunit of the T-cellreceptor.

It is a still further object of the present invention to providehybridization probes for identifying and isolating the T cell receptorgenes and subunits of the T cell receptor genes of other, non-mousespecies, including the human specie.

Another object of the present invention is to provide the protein oramino acid sequence of a murine alloreactive cytotoxic T lymptocytereceptor, including both of its subunits.

It is yet another object of the present invention to provide specificantibodies to the T cell receptor and subunits of the T cell receptorwhich are useful for identification, isolation, and in other methods forwhich antibodies are useful such as in delivering antibody bound drugsto a specific cell.

SUMMARY OF THE INVENTION

The complete nucleotide and amino acid sequences for the T cell receptorderived from the alloreactive cytotoxic T lymphocyte (CTL) clone 2C, ofBALB.B origin and specific for the product of a Class I gene at the Dend of the BALB/c H-2 complex (d haplotype) is disclosed. Two relatedbut distinct cDNA clones corresponding to the alpha and beta subunits ofthe T cell receptor were cloned and sequenced. The genes correspondingto these cDNA are rearranged and expressed in CTL clones but not inmyelomas and the rearrangement pattern varies with the CTL clone. Thecorresponding poly(A)⁺ RNA is composed of 5'-variable and 3'-constantregions. The encoded subunits are composed of a signal peptide, twoimmunoglobulin-like domains, each with one disulphide loop, atransmembrane peptide, and a cytoplasmic peptide. The two domains arehomologous to variable and constant domains of immunoglobulin chains,particularly those of lambda light chains. Besides having two cysteineresidues in each domain, the encoded subunits have a fifth cysteineresidue in the region between the disulphide bonded peptide loop of theconstant domain and the transmembrane peptide, at a position where thetwo subunits may be disulphide bonded to each other.

The constant region sequence of the beta subunit as encoded by the genepHDS11 from a cytotoxic T-cell is compared to the constant regionsequence of the beta subunit of a T helper cell and found to correspond.It is therefore concluded that the disclosed T cell specific cDNAsequences for the constant region of at least the beta subunit andprobably the alpha subunit may be used to produce protein, and antibodyto that protein, which are useful in isolation, identification, andother methods for both cytotoxic T lymphocytes and T helper cells.Expected homology among mammalian species, including man, furtherextends possible applications of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a Southern blot analyses, according to the method taught inMolec. Biol. 98, 503-517 (1975), of DNA from BALB/c embryo, myelomasMOPC315 and MOPC460, both BALB/c derived, and CTL lines 2C, G4, 1.5.2and 2.1.1, all BALB.B derived, and described by Kranz et al. in Proc.Nat. Acad. Sci. USA, 81, 573-577 (1984). DNA was digested with theindicated restriction enzymes, electrophoresed through 0.8% agarosegels, blotted onto nitrocellulose and hybridized with the ³² p-labelled,nick-translated insert from clone pHDS58 (FIG. 1a) and pHDS11 (FIG. 1b).Hybridization was carried out at 42° C. in 50% formamide and 5×SSC. Thefilters were washed at 65° C. in 0.2×SSC. Separate experiments showedthat BALB/c and BALB.B embryo patterns are indistinguishable. Numbers onthe left-hand side are M_(r) markers.

FIG. 2a, FIG. 2b and FIG. 2c are RNA blot analyses, according to themethod taught in Meth. Enzym. 100, 255-266 (1983), of poly(A)⁺ RNA fromvarious B- and T-cell lines. Probes: FIG. a, total insert of pHDS58;FIG. b, HpaII fragment containing 270 bp at the end of the pHDS58 insert(V-region probe); and FIG. c, total insert of pHDS11. RNA was extractedfrom B-cell lymphomas A20-2J and CHl and alloreactive (H-2^(b)anti-H-2^(d)) cytotoxic T-lymphocyte clones 2.1.1, 1.5.2, G4 and 2C.Approximately 1.5 microgram of poly(A)⁺ RNA was denatured with glyoxaland electrophoresed through 1% agarose gel in 10 mM sodium phosphatebuffer (pH 6.5). RNA was transferred to nitrocellulose membrane andhybridized to ³² p-labelled nick-translated probe DNAs, as described inFIG. 1. The filter used for hybridization with the first probe waswashed by boiling at 100° C. for 5 min in H₂ O and reused forhybridization with the second probe.

FIG. 3a and b are restriction maps of the insert of cDNA clones pHDS58(FIG. 3a) and pHDS11 (FIG. 3b). The map was constructed by a combinationof single and double restriction enzyme digestions of the plasmid DNA.The V, J, C and 5'-or 3'-untranslated regions are shown by shading,black, hatching and white, respectively. The sequencing strategy used todetermine the nucleotide sequence shown in FIG. 4 is shown. The arrowsindicate the directions and the extent of sequence determination. Theopen and closed circles indicate that the ends of the DNA fragmentslabelled are 5' and 3', respectively.

FIG. 4 are the nucleotide and predicted amino acid sequences of the cDNAclones pHDS58 (FIG. 4a) and pHDS11 (FIG. 4b). The nucleotide sequencewas determined by the method of Maxam and Gilbert, described in Meth.Enzym. 65, 499-560 (1980), according to the strategy shown in FIG. 3.Numbers given above the amino acid sequence designate amino acid residuepositions. The V, J, C, TM (transmembrane) and CY (cytoplasmic) regionsare indicated by horizontal arrows although exact boundaries areambiguous. Cysteine residues thought to be involved in intra-domain orinter-chain disulphide linkages are encircled. The potentialN-glycosylation sites (N-X-S or N-X-T) are also indicated.

FIG. 5 is a comparison of the V and C regions of the predicted aminoacid sequence of pHDS58 and the V and C regions, respectively, of thepredicted or determined amino acid sequences of five other polypeptidechains: a chain encoded by pHDS4/203, the beta-chain encoded by pHDS11,93G7 _(gamma) 1 immunoglobulin heavy chain, MOPC603 _(kappa) lightchain, and MOPC104E _(lambda) 1 light chain, as taught by Kabat et al.in Sequences of Proteins of Immunological Interest, (NIH, Bethesda(1983). Those residues common between the PHDS58 protein and any one ofthe other five chains are boxed. *, the residues common among all sixchains. Approximate boundaries of framework and hypervariable regions asthey appear in immunoglobulin V regions are indicated by horizontalarrows. The amino acid positions refer to those of PHDS58 protein.

FIG. 6a is a comparison of the J region amino acid sequences of pHDS58,that of pHDS4/203, as taught in Nature 309, 757-762 (1984), andconsensus sequences of the T-cell antigen receptor beta-chain, as taughtby Gascoigne et al. in Nature 309, 387-391 (1984), and immunoglobulinJ_(H), J_(kappa) and J_(lambda), as taught by Kabat et al. in Sequencesof Proteins of Immunological Interest (NIH, Bethesda (1983). Thoseresidues common between pHDS58 and any one of the other five J segmentsare boxed and shaded.

FIG. 6b is a comparison of the J region sequence of the amino acidsequence of pHDS4/203, pHDS11, 86T1, and Ig J_(H), J_(kappa), andJ_(lambda) consensus sequences. Those residues common between pHDS4/203J and any one of the other 5 J regions are boxed in and shaded.

FIG. 7 shows the proposed overall structure of the 2C T-cell receptor.Each receptor molecule is made up of two chains, alpha and beta, eachwith two extracellular Ig-like domains, an amino-terminal variable and acarboxy-terminal constant domain. Each of these domains is stabilized byan S--S bond between cysteine residues. The two chains are held by asingle interchain S--S bond located close to the cell's outer membrane.The protein is anchored on the membrane by two (one for each chain)hydrophobic transmembrane peptides. A short carboxy-terminal peptiderich in cationic residues extends into the cytoplasm in each chain. Bothalpha and beta chains are N-glycosylated, with the sites indicated onthe alpha subunit by CHO.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The preferred embodiment of the present invention is the structure andamino acid and nucleotide sequences of a heterodimeric glycoproteinwhich functions as a receptor on the surface of a T lymphocyte andcomprises both an alpha and a beta subunit. The invention also includescDNA hybridization probes and antibodies for the identification andisolation of T cell receptors from other species as well as fromcytotoxic and helper T cells. The T cell receptor and the nucleotide andamino acid sequences required to produce the protein or its componentsare defined by their substantial identity to the proposed tertiarystructure and nucleotide and amino acid sequences of the receptor of amurine alloreactive cytotoxic T lymphocyte.

The T-cell receptor is made up of two chains, each with twoextracellular Ig-like domains, an amino-terminal variable domain, and acarboxy-terminal constant domain. Each of these domains is stabilized bya disulfide bond between cysteine residues that are separated in thelinear sequence by 50-70 residues. The alpha chain, consisting ofapproximately 248 amino acid residues, has cysteine residues at aminoacid residues 22, 90 (variable region), 134, 184 (constant region), and202 (constant region adjacent transmembrane region). The beta chain,consisting of 282 amino acid residues, has cysteine residues at aminoacid residues 23, 91 (variable region), 140, 201 (constant region) and236 (constant region adjacent transmembrane region). The variable regionof the alpha subunit, consisting of amino acids 1-98, is joined to theconstant region, consisting of amino acids 112 to 222, by amino acids99-111. The variable region of the beta subunit, consisting of aminoacids 1-96, is joined to the constant region, consisting of amino acids110 to 254, by amino acids 97-109. Beyond the constant domain, eachsubunit has at its carboxyl-end a peptide carrying an extra cysteine, ahydrophobic stretch of about 20-22 amino acids followed by a shortstretch of approximately 5 amino acid residues in which cationicresidues abound. These segments correspond, respectively, to thetransmembrane and cytoplasmic domains characteristically found intransmembrane proteins.

These regions are indicated in FIG. 4a and FIG. 4b by horizontal arrows.The exact boundaries of a few adjacent regions are somewhat uncertain.

The alpha chain, as described, consists of 248 amino acids and has acalculated relative molecular mass of approximately 28,000. The chaincontains 23 negatively charged amino acid residues (15 aspartic acid and8 glutamic acid residues), and 19 positively charged amino acid residues(6 arginine and 13 lysine residues), corresponding to an isoelectricpoint near neutrality in the absence of post-translation modifications.There are four (4) potential sites for N-glycosylation, represented bythe tripeptide Asn-X-Ser/Thr. The potential sites for N-glycosylationare located at amino acid residues 70-72, 178-180, 142-144, and 221-223.

The alpha chain is preceeded by a sequence of 20 amino acid residues.This probably corresponds to a signal peptide, since most immunoglobulinchains carry a signal peptide 19-22 residues long. It has been shown byS. Schlossman that the N-terminal residue of the alpha subunit of theT-cell receptor is blocked, probably by a cyclized glutamine residue.The beta chain is preceeded by a sequence of about a dozen highlyhydrophobic residues that also probably comprises part of a signalpeptide.

The variable and constant regions of both chains are coded for bydistinct gene segments. The same gene appears to code for at least thebeta chain constant region of cytotoxic T-lymphocytes and the beta chainconstant region of helper T cells. The variable regions of cytotoxicT-lymphocytes (CTL) and helper T cells are probably encoded bynon-overlapping gene segments. The apparent homology between the aminoacid sequences of constant regions of both helper T cells and cytotoxicT cells enables one to use hybridization probes and antibodies directedagainst the constant region of the beta chain of the T receptor ofeither cell type to identify and isolate the T receptor of the othercell type. It is probable that the constant region of the alpha chain isalso substantially identical for helper T cells and CTLs.

The invention will be further understood from the following non-limitingexample wherein the amino acid and nucleotide sequences andhybridization probes for an alloreactive CTL clone are provided. All ofthe starting materials for this procedure are readily available to thoseskilled in the art from commercial or other sources.

ISOLATION OF T CELL-SPECIFIC CDNA CLONES

T cell-specific cDNA clones were isolated from the alloreactive CTLclone 2C, of BALB.B (mouse) origin and specific for the D end of theBALB/c H-2 complex (d haplotype). This clone was described by D. M.Kranz, D. H. Sherman, M. V. Sitkovsky, M. S. Pasternack, and H. N. Eisenin Proc. Natl. Acad Sci U.S.A., 81, 573-577 (1984).

The cDNA (complementary DNA) synthesized on the poly(A)⁺ RNA from 2C wassubtracted twice with poly(A)⁺ RNA from a mouse B cell lymphoma, A20-2J,according to the method of Hedrick et al. described in Nature 308,149-153 (1984). The B cell lymphoma, described by D. J. McKean, A. J.Infante, A. Nilson, M. Kimoto, C. G. Fathman, E. Walker and N. Warner inJ. Exp. Med., 154, 1419-1431 (1981) was used to remove cDNA that crossreacted with B lymphocytes rather than being specific for T lymphocytes.The method of Hedrick et al. is as follows:

(1) ³² p-labelled cDNA is synthesized from cytoplasmic poly(A)⁺ RNA of aT cell using oligo (dT) and reverse transcriptase;

(2) the RNA template is depleted by base hydrolysis;

(3) the cDNA is hybridized with B-cell mRNA such as B-cell lymphoma linepoly(A)⁺ RNA from Bal 12 or MBT_(H) -B; and

(4) unbound cDNA is removed by hydroxyapatite chromatography as T-cellspecific cDNA.

A library of cDNA from the CTL clone 2C was constructed from thesubtracted cDNA using the vector pBR 322 and a standard dC.dG tailingmethod, such as the one described by T. Maniatis, E. F. Fritsch, J.Sambrook in Molecular Cloning, A Laboratory Manual, p. 218, 269-307(Cold Spring Harbor Laboratory, 1982). The library was screened usingtwo hybridization probes. The first was the 2C cDNA prepared from thepoly(A)⁺ RNA of membrane-bound polysomes followed by subtraction withpoly(A)⁺ RNA from a second B-cell lymphoma, CHl, described by M. A.Lynes, L. L. Lanier, G. F. Babcock, P. J. Wettstein, and G. Haughton inJ. Immunol., 121:2352-2357 (1978). The second was the cDNA prepared fromthe total poly(A)⁺ RNA from the B-cell lymphoma A20-2J.

After screening about 20,000 independent transformants, a total of 140putative T-cell-specific cDNA clones were identified and grouped on thebasis of the corresponding genes, using the following procedures. First,plasmid DNA preparations from each of 20 randomly chosen cDNA cloneswere used as hybridization probes in RNA blotting analysis of 2Cpoly(A)⁺ RNA and DNA blotting analysis of 2C and embryo DNA. Thehybridization patterns allowed assignment of these clones to 12 groups.Second, mouse DNA inserts were dissected from a representative cDNAclone of each group and used as probes for colony hybridization of allof the cDNA clones. This procedure allowed assignment of 105 clonesamong the 12 groups, as shown in Table 1. The above procedures wererepeated for some of the remaining unassigned clones.

                  TABLE 1                                                         ______________________________________                                        T-cell-specific clones from a subtracted CTL cDNA library                             Size of                   Represent-                                          mRNA    No. of    Rearrange-                                                                            ative                                       Group   (kb)    clones    ment    clone                                       ______________________________________                                        T(thyI) 2.0     19        No      pHDS1                                       B       0.8     19        No                                                  C(beta) 1.4     18        Yes     pHDS11                                      F       1.7     13        No                                                  E       0.8     12        No                                                  D       1.7     9         Yes     pHDS58                                      K       1.3/1.1 9         No                                                  G       1.2     8         No                                                  A       1.5     4         Yes       pHDS4/203                                 P       2.3     2         No                                                  Q       n.d.    2         No                                                  I       2.2     1         No                                                  J       1.0     1         No                                                  L       1.5     1         No                                                  N       4.4     1         No                                                  R       0.8     1         Maybe   pHDS86                                      Total           120                                                           ______________________________________                                    

The 2C-specific cDNA clones were identified and their plasmid DNA usedas hybridization probes in a series of RNA blotting experiments in orderto confirm the T cell-specific expression of the corresponding genes.The T cell-specific cDNA were grouped into sets according to the sizesof the corresponding mRNA present in 2C. One probe was found to be forthe T cell specific cell surface marker Thy-1 on the basis of itshybridization to a previously identified Thy-1 cDNA clone provided byMark Davis.

IDENTIFICATION OF TWO DISTINCT CLASSES OF T CELL-SPECIFIC CDNA CLONESWHOSE GENES ARE REARRANGED IN CTL'S

Representative cDNA clones of each set of T cell-specific cDNA groupedaccording to its size were used as hybridization probes. Comparison ofthis cDNA with EcoRI-digested genomic DNA from 2C and BALB.B embryos bythe Southern gel blotting method led to identification of three distinctclasses of cDNA, represented by clone pHDS11, clones pHDS4 and pHDS203,and clone pHDS58.

FIG. 1a shows the results of Southern gel blotting analysis using clonepHDS58 as the hybridization probe with DNA from BALB/c embryos, four CTLclones of different specificities and two myelomas digested with KpnI,EcoRI, and BG1II restriction enzymes. With each of the three enzymes,the patterns obtained with 2C DNA are different from the patternsobtained with BALB embryo DNA. Clone 2C showed an extra fragment notpresent in embryo DNA and lacked fragments which were present in embryoDNA. The myeloma DNA patterns are the same as the embryo DNA patterns.

FIG. 1b shows the results of Southern gel blotting analysis using clonepHDS11 as the hybridization probe with DNA from BALB/C embryos, myelomaP3 (BALB/C derived), and CTL 2C (BALB.B) digested with PvuII and EcoRI.

These results strongly suggest that the genes corresponding to clonepHDS58 and clone pHDS11 are rearranged in cytotoxic T cells but not inmyelomas and are clearly distinct.

The expression of the pHDS58 gene in CTL clones and B lymphomas wasexamined by Northern hybridization analyses according to the method ofP. S. Thomas in Meth. Enzym., 100, 255-266 (1983). As shown in FIG. 2aand FIG. 2c, when the whole insert of pHDS58 (FIG. 2a) or pHDS11 (FIG.2c) was used as the hybridization probe, poly(A)⁺ RNA of distinct sizeswas detected in clone 2C and three other independently derivedalloreactive CTL clones but not in either of the B lymphomas A20-2J andCHl.

When pHDS58 was used as the hybridization probe, the size of the majorRNA species in 2C (1.7 kliobases, kb) was slightly greater than in theother three CTL clones (1.6 kb). Minor RNA species of about 1.2 kb weredetected in all four CTL clones.

When a DNA fragment containing 270 base pairs from the 5' end of thepHDS58 insert was used as the hybridization probe, only the 1.7 kb RNAspecies of 2C was detected and the other three CTL clones and the B-celllymphomas exhibited no hybridization, as shown by FIG. 2b. FIG. 3a showsthe region of the cDNA sequence covered by the probe. These resultssuggest that the 1.6-1.7 kb RNA is composed of a common 3' sequence (Cregion) and a CTL clone-specific 5' sequence (V region), as had beenseen with immunoglobulin and the T cell receptor beta chain.

When pHDS11 was used as the hybridization probe, the size of the RNA wassomewhat variable from one CTL to another. CTL 2C, 1.5.2, and 2.1.1 allgave a major RNA component of about 1,300 bases while CTL G4 containedtwo major components of 1,400 and 1,200 bases.

NUCLEOTIDE SEQUENCE ANALYSES

As shown in FIG. 3, restriction maps of the pHDS58 cDNA clone (FIG. 3a)and the pHDS11 cDNA clone (FIG. 3b) were constructed using standardprocedures such as the ones described by T. Maniatis, E. F. Fritsch, J.Sambrook in Molecular Cloning, A Laboratory Manual, p. 3-54, 374-401(Cold Spring Harbor Laboratory 1982). The DNA sequence was determinedusing the method of A. M. Maxam and W. Gilbert in Methods in Enzymology,L. Grossman and V. Moldave, Editors, 65:499-560 (Academic Press, N.Y.1980).

The entire nucleotide sequence of the 1,054 base pair insert of clonepHDS11 is shown in FIG. 4b. The longest open reading frame is composedof an 879 nucleoticle stretch whose corresponding amino acid sequence of293 residues is also shown in FIG. 4b. The codons are numbered startingwith the triplet GAC at nucleotide position 36-38. There is a stretch ofabout a dozen highly hydrophobic residues at the 5' end of the openreading frame that probably comprise part of a signal peptide. Homologybetween the pHDS11 protein and immunoglobulin variable regions (V),particularly V_(k) regions, suggests that Asp at position 1 is theN-terminal residue. The variable region is between codons 1 and 96 andthe joining region from codons 97 to 109.

The major body of the constant region is defined by codons 110 (Glu) and236 (Cys). Codon 236 (Cys) is prior to the N-terminus of thetransmembrane segment (TM) which extends from codon 256 to 277. Theconstant region pHDS11 sequence is identical to the sequence of thecorresponding region of the thymocyte cDNA clone, 86T1, described byHedrick et al., except for one base pair in codon 159.

The sequence identity of the constant region between pHDS11 and 2B4#71from a cDNA clone isolated from a T helper cell (T_(H)) hybridomaspecific for anti pigeon cytochrome C is also striking. The T_(H) cDNAclone is described by Yih Chien, N. R. J. Gascoigne, J. Kevaler, N. E.Lee, and M. M. Davis in Nature 309, 322-326 (1984) and Nature 310,387-391 (1984).

The two sequences are identical throughout the constant regions and theentire 3' untranslated region except for two base pair differences atcodon 159 and at nucleotide positions 992.

The joining region from codons 97 to 109 in the pHDS11 sequence isdistinct from 8671 and 2B4#71, but homologous to the joining segments ofIg genes. As shown in FIG. 6b, the pHDS11 joining sequence correspondsexactly to the sequence of the J_(T) ⁷ genomic segment recentlyidentified and characterized by Chien et al.

The pHDS11 sequence between codons 1 and 96 is quite different from thecorresponding region of either 86T1 or 2B4#71. There are areas ofconserved residues between the three sequences, however, and betweenthese sequences and the immunoglobulin variable regions of both heavyand light chains, in particular, the two cysteine residues involved inintradomain disulfide linkages and the Trp residue at residue 34.

Another stretch of a highly conserved hydrophobic region of about 22residues, immediately between the C-terminal five hydrophilic residuesand the constant region constitutes a transmembrane (TM) peptide. Thefive hydrophilic residues are thought to extend into the cytoplasm.

Overall, the gene defined by pHDS11 can encode a processed protein of282 residues with a relative molecular mass of 33,000 daltons. There arefour potential sites for N-glycosylation.

The entire nucleotide sequence of the 1,372 base pair insert of clonepHDS58 is shown in FIG. 4a. The longest open reading frame begins withthe Met codon at nucleotide positions 56-58, extends over a stretch of804 bp, and ends at nucleotide position 859. The corresponding aminoacid sequence of 248 residues is also shown in FIG. 4a. The codons andamino acids are numbered starting with the triplet CAG (Gln) atnucleotide positions 116-118. The amino acid sequence immediatelyfollowing the Met and extending to the Gln is highly hydrophobic andprobably comprises part of a signal peptide.

After the Gln, the sequence of clone pHDS58 is significantly homologousto that of pHDS11 and the polypeptide chain encoded by pHDS4/203, aswell as to the sequences of an immunoglobulin heavy-chain (93G7), animmunoglobulin kappa light chain (MOPC603) and an immunoglobulin lambda1 light chain (MOPC104E).

The homology is shown in FIGS. 5a and 6a, where the pHDS58 amino acidsequence is compared with the V (or V+D), J and C region sequences ofthe other five polypeptide chains. Sequence homology is evident in allthree regions, but is most striking in the J region, where it amounts to38-62%. In the V regions, the pHDS58 sequence is related to the otherfive sequences by 22-29% homology. The six amino acid residues that areconserved in this region among pHDS11, pHDS4/203 and the three types ofimmunoglobulin chains (Gln, Cys, Trp, Gln, Tyr and Cys at positions 5,22, 34, 37, 88 and 90, respectively) are also shared by pHDS58.

The sequence homology is least in the C regions, where it ranges over12-20%. Nevertheless, the relatedness is evident around the two Cysresidues that form intra-domain disulphide bonds in immunoglobulinchains. Throughout the V, J and C regions, the pHDS58 sequence, like thepHDS11 and pHDS4/203 sequences, is more homolgous to the light chains(25% and 27% for kappa and lambda respectively) than to the heavy chain(23%).

Beyond the C region, the pHDS58 polypeptide chain exhibits no obvioussequence homology to the corresponding regions of pHDS11 or the chainencoded by pHDS4/203. Nevertheless, the three polypeptide chains areorganized in a very similiar fashion in these regions. Thus, as in thepHDS11 and pHDS4/203 chains, the C region of the pHDS58 chain isfollowed by a peptide carrying an extra cysteine, then by a stretch ofabout 20 hydrophobic residues that corresponds to a transmembranepeptide and finally by a short hydrophilic C-terminal peptide thatpresumably extrudes into the intracytoplasmic space.

In FIG. 4a, these regions of pHDS58 are indicated by horizontal arrows.The exact boundaries of a few adjacent regions are somewhat uncertain.For instance, it is not possible to determine unambigiously theN-terminus of the processed protein from the nucleotide sequence of thecDNA alone. However, the Gln at nucleotide positions 116-118 is the bestcandidate for the following reasons. First, the proposed assignmentplaces the first Cys residue at position 22, while the corresponding Cysresidues of most immunoglobulin chains are at positions 22 or 23.Second, most immunoglobulin chains carry a signal peptide of 19-22residues. The present assignment makes the proposed signal peptide 20residues long. Finally, the pHDS58 chain is an excellent candidate forthe alpha subunit of the T-cell receptor, whose N-terminal residue hasrecently been shown to be blocked probably by a cyclized glutamineresidue by S. Schlossman.

The proposed processed polypeptide chain is 248 amino acids long. Thecalculated relative molecular mass is approximately 28,000. The chaincontains 23 negatively charged (15 aspartic acid and 8 glutamic acidresidues) and 19 positively charged residues (6 arginine and 13 lysineresidues), corresponding to an isoelectic point near neutrality in theabsence of post-translation modifications. As shown in FIG. 4a, it hasfour potential sites for N-glycosylation. Endoglycosidase F digestion ofthe alpha/beta heterodimers of mouse helper T cells in a human T-celltumour by J. P. Allison suggested that both subunits of these T-cellreceptors are N-glycosylated. Typical N-glycosylation sites are shown bythe tripeptide Asn-X-Ser/Thr. Recent studies have also shown that thealpha subunit of CTL clone 2C is N-glycosylated. As shown in FIG. 4a,the proposed N-glycosylation sites are at amino acid residues 70-72,178-180, 192-194 and 221-223.

The variable region of the alpha subunit is between amino acids 1 and98. The major body of the constant region is defined by amino acids 112(Tyr) and 221 (Asn). A joining region between the variable region andthe constant region extends from amino acids 99 to 111. Amino acid 202(Cys) is prior to the N-terminus of the transmembrane segment (TM) whichextends from amino acids 222 to 243.

The joining region from amino acid residues 99 to 111 is homologous tothe joining segments of Ig genes. As shown in FIG. 6a, the pHDS58joining sequence corresponds to the sequences of the T-cell antigenreceptor beta-chain described by Gascoigne et al. in Nature 310, 387-391(1984), and immunoglobulin J_(H), J_(kappa), and J_(lambda) described byKabat et al. in Sequences of Proteins of Immunological Interest, (NIH,Bethesda (1983)).

As shown in FIG. 5a, the pHDS58 sequence between amino acids 1 and 100(variable region) and amino acids 110 and 200 (constant region) haveareas of conserved residues with a chain encoded by pHDS4/203, thebeta-chain encoded by pHDS11, 93G7_(gamma) 1 immunoglobulin heavy chain,MOPC603_(kappa) light chain, and MOPC 104E_(lambda) 1 light chain.

Another stretch of a highly conserved hydrophobic region of about 20residues, between the C-terminal five hydrophilic residues and theconstant region constitutes a transmembrane (TM) peptide. The fivehydrophilic residues are thought to extend into the cytoplasm.

Overall, the gene defined by pHDS58 can encode a processed polypeptidechain of 248 amino acid residues with a relative molecular mass ofapproximately 28,000 daltons. There are four potential sites forN-glycosylation.

SUMMARY

On the basis of T cell-specific expression, T cell-specificrearrangement, and sequence homology to immunoglobulin chains and to thebeta chain purified by Reinherz et al. (personal communication) from a Tcell tumor REX and the cDNA clones reported by Hedrick et al. (TM 186)and Yanagi et al. (YT35), pHDS11 has been shown to code for the betasubunit of the mouse T cell receptor.

The gene defined by cDNA clone pHDS58 is expressed and rearrangedspecifically in T cells. This gene and its product share severalcharacteristics with pHDS11 and pHDS4/203, and their products. Thesesimiliarities may be summarized as follows.

1. The genes are specifically expressed in various CTL clones but not inB lymphomas.

2. The genes are rearranged in CTL clones but not in myelomas, and therearrangement pattern varies with CTL clone.

3. The corresponding poly(A)⁺ RNA is composed of 5'-variable and3'-constant regions.

4. The primary sequences suggests that the encoded protein is composedof a signal peptide, two immunoglobulin-like domains, each with onedisulphide loop, a transmembrane peptide and a cytoplasmic peptide.

5. The two domains are homologous to the V and C domains ofimmunoglobulin chains, particularly those of lambda light chains.

6. Besides having two cysteine residues in each domain, the encodedprotein has a fifth cysteine residue in the region between the C domainand the transmembrane peptide, at a position where the chain might bedislphide bonded to another chain.

The proposed structure for the T cell receptor is show in FIG. 7. Thereceptor molecule is made up of two chains, each with extracellularimmunoglobulin-like domains, an amino-terminal variable domain and acarboxy-terminal constant domain. Each domain is stabilized by adisulfide bond between cysteine residues that are separated by a linearsequence of 50-70 residues.

Cysteine residues at positions 202 and 236 of the alpha and betasubunits, respectively, form a single, interchain disulfide bond locatedclose to the cell outer membrane. This bond may link the two subunits inthe intact molecule and account for the difference in apparent molecularweight between the unreduced and reduced receptor (90,000 daltons versus40-45,000 daltons) in SDS-polyacrylamide gel electrophoresis. Themolecular weights of the alpha and beta subunits are each less by about10,000 to 15,000 daltons than the apparent molecular weight observed inSDS-polyacrylamide gel electrophoresis. It is possible this differenceis due to N-glycosylation of the subunits.

A stable association between the two subunits may be required to form aneffective binding region, as is characteristic of immunoglobulins. Theobligate participation of two different subunits in the formation of asingle combining site means that combinational variability is likely tocontribute to structural and functional diversity of these receptors.

The presence of 18-22 hydrophobic amino acids, followed by a shortstretch of amino acid residues in which there are many cationicresidues, at the carboxyl-end of the constant domain corresponds to thetransmembrane and cytoplasmic domains that are characteristically foundin transmembrane proteins.

The T cell receptor is defined in the example by its amino acidsequence, nucleotide sequence of variable, joining, constant,transmembrane, and cytoplasmic segments, arrangement of disulfide bondsand glycosylation sites.

Considering the high sequence homology observed between twocorresponding genes belonging to two mammalian species (for example,mouse Ig C_(k) and human Ig C_(k) are 60% homologous, mouse IgC_(lambda) and human Ig C_(lambda) are 65% homologous, rabbit C_(H2) andhuman C_(H2) are 63% homologous, and rabbit C_(H3) and human C_(H3) are66% homologous (Davis et al, Microbiology, 2nd Ed., p. 441 (Harper & RowPublishers, 1973) and comparing homology between the gene sequence ofHedrick et al and Yanagi et al for the beta subunit of the T cellreceptor) with that of pHDS11, it is highly probable that the alpha andbeta genes of different mammalian species, including human, can becloned from the T cell cDNA libraries of these species using thedisclosed mouse alpha and beta cDNA as hybridization probes.

The cDNA may be used in various systems known to those skilled in theart to make large quantities of the actual T cell receptor protein. Thisis of tremendous use since a major limitation on previous work was thesmall amount of T cell receptor available for study. Both procaryoticand eucaryotic systems are useful for production of T cell receptorprotein or the alpha or beta subunits of the receptor.

An example of a method of producing protein from the cDNA is taught bycopending application U.S. Ser. No. 592,231, to Gillies et al. now U.S.Pat. No. 4,663,281, entitled "Enhanced Production of ProteinaceousMaterials in Eucaryotic Cells" and filed Mar. 22, 1984.

Another method is taught by Gray and Goeddel, entitled "Cloning andExpression of Murine Immune Interferon cDNA", Proc. Natl. Acad. Sci.USA., 80:5842-5846 (1983).

The eucaryotic system has the advantage over the procaryotic system thatthe expressed molecule is glycosylated.

The T cell receptor protein or polypeptide sequences may be used forfurther studies, in systems for the detection of anti-T cell antibodies,and in other procedures known to those skilled in the art. They may alsobe used for the production of specific T cell receptor antibodies whichare directed against the entire molecule, the alpha or beta subunit, orspecific regions within the molecule, such as the constant region. Theantibodies directed against the constant region of the cytotoxic Tlymphocyte should be equally effective against the constant region of Thelper cells.

Antibodies may be produced from the protein or its subunits usingconventional techniques known to those skilled in the art. Nucleotidesequences may also be utilized to produce short peptides or fusionproteins which are then bound to a carrier protein such as bovine gammaglobulin (BTG) for injection into an animal for the production ofantibodies. For example, an animal may be immunized against the proteinor peptide bound protein and immunoglobulin isolated from the serum. TheB cells from the immunized animal with the desired specificity may alsobe fused with a cell line which is maintained in cell culture, such asmyelomas or other tumor cell lines, to form hybridomas for thecontinuous production of antibody.

Antibodies are useful in isolation procedures such as by affinitychromatography wherein antibody is bound to a solid matrix and byprecipitation of soluble antibodies in solution. Antibodies are usefulin analysis and identification using any of a number of well knowntechniques. A recently developed use of antibodies involves binding anagent, such as a drug, to the antibody, then injecting the boundantibody into a patient so that the drug is delivered only to thedesired site. In the present invention, an example of such a use wouldbe delivery of a chemotherapeutic compound to malignant T cells in apatient with a T cell lymphoma.

The invention may be embodied in other specific forms without departingfrom the spirit and scope thereof. These and other modifications of theinvention will occur to those skilled in the art. Such other embodimentsand modifications are intended to fall within the scope of the appendedclaims.

What is claimed is:
 1. An antibody specific for regions of the alphasubunit of a mammalian T lymphocyte receptor or an antigenic portionthereof, present on more than one clone of cells bearing T lymphocytereceptors.
 2. The antibody of claim 1 wherein the antibody is directedagainst the constant region of the alpha subunit.
 3. The antibody ofclaim 1 wherein the antibody is directed against the joining region ofthe alpha subunit.
 4. The antibody of claim 1 wherein the antibody isdirected against the transmembrane region of the alpha subunit.
 5. Theantibody of claim 1 wherein the T lymphocyte receptor is a clonallydiversified integral membrane glycoprotein comprising an alpha subunitand a beta subunit, said glycoprotein of mammalian origin functioning asa receptor for antigens and major histocompatibility complex geneproducts when located on the surface of the T lymphocyte, wherein saidsubunits comprise:a signal peptide of between 12 and 22 amino acidresidues; a variable, immunoglobulin-like domain having a binding sitespecific for at least one MHC gene product, wherein said variable domainhas a first and second cysteine residue from the N-terminus; a constant,immunoglobulin-like domain having substantial identity to acorresponding amino acid sequence in receptors of both helper Tlymphocytes and cytotoxic T lymphocytes, wherein said constant domainhas a third and a fourth cysteine from the N-terminus; a joining regionincluding an amino acid sequence connecting said variable and saidconstant domains; a transmembrane peptide including a sequence ofpredominantly hydrophobic amino acids; and a short cytoplasmic peptideincluding a sequence of predominately cationic amino acids; wherein saidtransmembrane peptide is between said constant domain and saidcytoplasmic region, and wherein the constant region between said fourthcysteine and said transmembrane peptide has a fifth cysteine residue forformation of a disulfide bond between said alpha subunit and said betasubunit, and wherein each of said subunits contain four sites forN-glycosylation, said sites located at a first site between said firstcysteine and said second cysteine, at a second site between said thirdcysteine and said fourth cysteine, and at a third and fourth site in theregion between said fourth cysteine residue and said transmembranepeptide when said protein is produced by an alloreactive cytotoxic Tlymphocyte clone 2C of BALB.B origin and specific for the D end of theBALB/c H-2 complex (d haplotype).
 6. The antibody of claim 1 specificfor a polypeptide encoded by a nucleotide sequence having sufficienthomology to a cDNA sequence encoding the constant region of the alphasubunit of a T lymphocyte receptor of mammalian origin to be isolated byhybridization and comprising the amino acid sequence: C L F T D F D S QI N V P K T M E S G T F I T D K T V L D M K A M D S K S N G A I A W S NQ T S F T C.